Summary: During the past year I have finished work that describes the gene inversion that is responsible for hemophilia A in a dog colony in Chapel Hill, North Carolina. The key features of this animal model are that the factor VIII levels are undetectable and the bleeding phenotype is identical to that of humans with severe hemophilia A (e.g., spontaneous bleeding in joints and soft tissues, prolonged bleeding with trauma and surgery, and prolonged in vitro clotting times of plasma). The key findings of my research were that the hemophilia A dog colony in Chapel Hill has an inversion that is identical in mechanism to a common mutation seen in humans with hemophilia A. Further, the local sequence of DNA is one which may be amenable to correction by mRNA splicing, and is identical to that found in humans. This animal model could therefore be used to test mRNA splicing constructs that could be used in humans to correct the common inversion that causes hemophilia A. During the past year I have published with my CBER collaborators and external collaborators the results of studies of the effects of adenovirus vectors on non-human primates. These results indicate dose-dependent toxicity which manifests as abnormalities of the coagulation system, which include abnormalities of fibrinogen, prolongation of clotting times, and thrombocytopenia. In addition, there are increases in high-molecular weight von Willebrand factor multimers which may mediate the platelet consumption that takes place. The observation that these abnormalities occur with a relatively innocuous adenovirus vector (at high doses) suggests that this animal model might be studied to assess the beneficial effects of certain biologicals (particularly activated protein C) which have proven to be clinically useful for patients with bacterial sepsis and disseminated intravascular coagulation. If such agents could be shown to have a protective effect against these toxic effects seen with certain viral infections, then it may prove worthwhile to study these interactions to see if they might be applicable to therapy of certain viral infections characterized by DIC (e.g., dengue, ebola and other hemorrhage-inducing viruses). If data to this effect can be developed in the upcoming year, I may introduce a separate proposal to study these agents in the context of counter-bioterrorism efforts. During the last year I have begun a study whose aim is to map genetic loci in mice that control the immune response to human factor IX. As it turns out, there are inbred strains of mice (i.e., C57Bl/6J and A/J) that have dramatically different antibody responses to human factor IX when given as a protein (without adjuvant) or in the context of gene therapy vector delivery. The value of this observation is that there are inbred mice which are derived from these parental strains and which are mapped very extensively with regard to genetic polymorphisms found in the mouse genome. These well-characterized mice can be immunized with human factor IX and scored for the immune response. It is anticipated that this approach will permit mapping of any genetic loci that control the antibody response to factor IX. Once mapped any loci can be investigated for their relevance to the immune response to foreign proteins. If genetic factors that control the immune response in mice can be found, it would be straightforward to assess their possible contribution to inhibitor antibodies in humans with hemophilia B. The importance of this is that inhibitor antibodies are a well-known and feared complication of conventional therapy with clotting factor concentrates and a feared and for now hypothetical complication of gene therapy of hemophilia B. Other genetic approaches to the elimination of inhibitor antibodies includes the possibility that inhibition of gene expression by short, sequence specific, double stranded RNA oligonucleotides could be used to decrease the expression of unique antibodies (e.g., to clotting factors). At this point I have a small amount of toxicology data to suggest that there is not demonstrable toxicity to the liver or bone marrow in mice injected with these compounds. I intend to design certain of these compounds to test their ability to effect gene-specific inhibition of antibody expression in mice.